PROJECT SUMMARY Glioblastoma is the most common type of brain tumor and is currently incurable. The lack of effective treatments highlights the urgent need for identifying mechanism-based therapeutic approaches. Substantial experimental evidence has recently revealed a population of neural stem cell (NSC)-like glioma stem cells (GSCs) that possess an inexhaustible ability to self-renew as the ?root? of glioblastoma. Like NSCs, GSCs are known to maintain their stemness by interacting with niches, which provides proper cues to prevent them from differentiating. But how GSCs manage to sustain their self-renewal capacity in the sub-optimal environment outside the niches, particularly during the process of invasion and migration, remains less clear. As part of our effort to identify potential glioma suppressors involved in the regulation of central nervous system development, we discovered that RNA binding protein Quaking (QKI) is a major regulator of NSC and GSC self-renewal. QKI is frequently deleted or mutated in human glioblastomas. Using a newly established animal model, we genetically demonstrated that QKI is a bona fide glioma suppressor whose depletion strongly promotes gliomagenesis. Functionally, we revealed that QKI is a key regulator of cellular endocytosis that controls receptor trafficking, degradation, and signaling desensitization. Specifically, we showed that depletion of QKI led to the enrichment of cytoplasmic membrane-bound Wnt and Notch receptors (Frizzled and Notch1) and subsequent signal hyperactivation. Given that Wnt and Notch1 are two major signaling cascades involved in maintaining NSC and GSC stemness against differentiation, we propose that QKI modulates NSC and GSC self-renewal and gliomagenesis by controlling endolysosome-mediated Frizzled and Notch1 degradation. To test this hypothesis, in Aim 1, we will determine how QKI regulates the endolysosome-dependent degradation of Wnt receptor Frizzled in NSCs and GSCs. In Aim 2, we will delineate the molecular mechanism by which QKI modulates RNA alternative splicing of the endocytic regulator Numb and the endolysosomal Notch1 degradation. Together, these studies will elucidate the molecular mechanisms underlying QKI-mediated endolysosome-dependent regulation of Wnt and Notch1 signal activation, and more importantly, they will contribute to the development of therapeutic strategies that specifically target QKI-depleted glioblastoma.